Factors Affecting Transient Gene Expression in Apple Cotyledons following Particle Bombardment

Developing an efficient gene transfer system for apple (Malus ×domestica L.) remains a major objective in genetic engineering efforts of this fruit crop. Transient expression of the uidA gene coding for β-glucuronidase (GUS) and driven by the cauliflower mosaic virus 35S promoter (CaMV35S) has been induced in apple cotyledonary explants of mature seeds by tungsten particle bombardment using the Particle Inflow Gun (PIG). Several factors that affect transient expression of the GUS gene in apple cotyledons were investigated. The gene transfer efficiency was monitored by recording the number of blue spots observed on explants two days following bombardment. Precultivation of cotyledons for 18 hours before bombardment significantly increased the number of blue foci. Of the three different precipitation methods tested including water, 25% PEG, and 60% glycerol, the latter was the most effective for coating DNA onto tungsten particles. Washing DNA-coated tungsten particles with 70% ethanol and resuspending in 100% ethanol significantly enhanced gene delivery to cotyledons. The amount of particles used for each bombardment also influenced GUS expression. About 0.5 mg of particles per shot resulted in the highest number of blue foci. Using larger quantity of particles (i.e., 2 mg) drastically decreased GUS expression probably due to the toxicity of tungsten particles.

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Factors Affecting Transient Gene Expression in Mango Tissue Using Particle Bombardment

HortScience ◽

10.21273/hortsci.39.4.754e ◽

2004 ◽

Vol 39 (4) ◽

pp. 754E-755

Author(s):

Vaughan Agrez* ◽

Zora Singh ◽

Mis Gill

Keyword(s):

Gene Expression ◽

Particle Bombardment ◽

Mangifera Indica ◽

Transient Gene Expression ◽

Gus Expression ◽

35S Promoter ◽

Helium Pressure ◽

Factors Affecting ◽

Pre Treatment

Particle bombardment seems to be a promising method for genetic transformation of mango and the information on various factors affecting transient gene expression is inconclusive. Pro-embryonic masses (PEMs) of mango (Mangifera indica L. cv. Kensington Pride) used for particle bombardment were established and maintained in vitro using various liquid and semisolid media containing 2,4-D. Various factors affecting the biolistic transformation efficiency in mango and subsequent transient gene expression were optimized using β-glucuronidase (GUS) as a reporter gene driven by CaMV 35S promoter. Our results show that bombardment pressure significantly affects transient gene expression with the best results obtained from PEMs bombarded at 1200 kPa of helium pressure under vacuum. The application of osmoticum pre and post-bombardment also significantly increased the transient gene expression in the PEMs as compared to the controls. Mannitol (0.2 m) proved to be the best osmoticum in improving transient GUS expression as compared to sorbitol. The duration of exposure of PEMs to mannitol (0.2 m) both pre and post-bombardment also played a significant role in improving transient gene expression. The transient GUS expression was significantly highest with a pre-treatment of 0.2 m mannitol for 4 hours as compared to 0, 8 and 12 hours. The post-bombardment treatment of 0.2 m mannitol for 16 hours resulted in significantly highest transient gene expression as compared to 0, 4, 8, 12, 20 and 24 hours. In conclusion, PEMs of `Kensington Pride' mango bombarded at 1200 kPa, which were exposed to mannitol (0.2 m) for 4 and 16 hours pre and post bombardment respectively, resulted in the highest transient GUS expression (25.1 GUS foci/mg PEMs).

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Transient expression of foreign chimeric genes in the gymnosperm hybrid larch following electroporation

Canadian Journal of Botany ◽

10.1139/b91-220 ◽

1991 ◽

Vol 69 (8) ◽

pp. 1731-1736 ◽

Cited By ~ 13

Author(s):

Pierre J. Charest ◽

Yvonne Devantier ◽

Christine Ward ◽

Catherine Jones ◽

Ulriche Schaffer ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Expression System ◽

Transient Gene Expression ◽

Inducible Promoter ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Hybrid Larch ◽

Naked Plasmid ◽

Angiosperm Species

A transient gene expression system using electroporation and naked plasmid DNA has been developed for hybrid larch (Larix × eurolepis). The β-glucuronidase, neomycin phosphotransferase II, and chloramphenicol acetyltransferase genes were used effectively, but the latter was found to be the most useful. Electroporation conditions were comparable with protocols developed for other conifer and angiosperm species. Of the parameters tested the optimum conditions were 300 V, 150 μF, and 300 μg/mL pCaMVCN DNA. The 35S promoter of cauliflower mosaic virus yielded a stronger level of transient expression than the nopaline synthase promoter, which is consistent with other studies. A construct with the wound-inducible promoter of the potato proteinase IIK gene and the chloramphenicol acetyl transferase reporter coding sequence did not yield to any transient gene expression, even after induction with acetylsalicylic acid and exposure to ultraviolet radiation. Key words: larch, Larix × eurolepis, electroporation, transient gene expression.

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Particle bombardment – mediated gene transfer and GFP transient expression in Setaria viridis

Plant Signaling & Behavior ◽

10.1080/15592324.2018.1441657 ◽

2018 ◽

Vol 13 (4) ◽

pp. e1441657 ◽

Cited By ~ 4

Author(s):

Muruganantham Mookkan

Keyword(s):

Gene Transfer ◽

Transient Expression ◽

Particle Bombardment ◽

Setaria Viridis

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Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Physiologia Plantarum ◽

10.1111/j.1399-3054.1996.tb00236.x ◽

1996 ◽

Vol 96 (4) ◽

pp. 630-636 ◽

Cited By ~ 10

Author(s):

Manuel Rey ◽

Maria Victoria Gonzalez ◽

Ricardo J. Ordas ◽

Raffaela Tavazza ◽

Giorgio Ancora

Keyword(s):

Gene Expression ◽

Particle Bombardment ◽

Transient Gene Expression ◽

Radiata Pine ◽

Factors Affecting

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Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132003000100001 ◽

2003 ◽

Vol 46 (1) ◽

pp. 1-6 ◽

Cited By ~ 17

Author(s):

João C. Bespalhok Filho ◽

Adilson K. Kobayashi ◽

Luiz F. P. Pereira ◽

Rafaelo M. Galvão ◽

Luiz G. E. Vieira

Keyword(s):

Gene Expression ◽

Particle Bombardment ◽

Citrus Sinensis ◽

Sweet Orange ◽

Transient Gene Expression ◽

Gus Expression ◽

Poncirus Trifoliata ◽

Helium Pressure ◽

High Osmolarity ◽

Carrizo Citrange

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis).

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MOLECULAR CHARACTERIZATION AND TRANSIENT EXPRESSION OF MAIZE SUS1 PROMOTER

HortScience ◽

10.21273/hortsci.29.4.253f ◽

1994 ◽

Vol 29 (4) ◽

pp. 253f-254

Author(s):

Xiao-Fang Huang ◽

Binh Nguyen-Quoc ◽

Serge Yelle

Keyword(s):

Transient Expression ◽

Gus Expression ◽

35S Promoter ◽

Plant Tissues ◽

Upstream Sequence ◽

Specific Expression ◽

Gene Sequence Analysis ◽

Sequence Elements ◽

Young Leaves

Sucrose synthase (SS) is one of the key enzymes in plant carbohydrate metabolism. In maize, this enzyme is encoded by two genes, Sh1 and Sus1. We have isolated and determined the 5'-upstream sequence of maize Sus1 gene and compared it with the corresponding sequence in Sh1 gene. Sequence analysis revealed that there was a weak homology between the two promoters and no common sequence elements were found. To understand the differential regulation of the expression of the two genes, we constructed chimeric GUS fusions using the two promoters of SS genes. By using the biolistic system, we delivered these constructs into various plant tissues, and their transient expression was studied. Our results showed that the two promoters of SS genes directed tissue-specific expression in the same way that the two genes are expressed in vivo. The effectiveness of the expression of the constructs was recorded by counting the total blue expression units (blue spots) per shot and by fluorometric assays. High levels of GUS activity were detected in the immature embryos, young coleoptiles, and heterotrophic young leaves bombarded with the Sus–GUS construct. More than 100 expression units were observed in these tissues. Compared with the transient expression of the 35S promoter in the same tissue, Sus promoter activity was twice as high. Strong Sus–GUS expression was also detected in the aleurone cells of developing kernels. In contrast, the Sh-GUS construct was expressed only in the endosperm with an activity twice as high as that of Sus–GUS and 35S–GUS in the same tissue. The results will be discussed in terms of the physiological roles of the two SS isozymes in plant tissues.

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Research note: The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

Functional Plant Biology ◽

10.1071/pp01121 ◽

2002 ◽

Vol 29 (5) ◽

pp. 649 ◽

Cited By ~ 31

Author(s):

Aziz Smouni ◽

Laurent Laplaze ◽

Didier Bogusz ◽

Fathia Guermache ◽

Florence Auguy ◽

...

Keyword(s):

Vascular Tissue ◽

Gus Expression ◽

Cauliflower Mosaic Virus ◽

Camv 35S Promoter ◽

35S Promoter ◽

Casuarina Glauca ◽

Camv 35S ◽

Highly Active ◽

One Year

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

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Grapevine Protoplasts as a Transient Expression System for Comparison of Stilbene Synthase Genes Containing cGMP-Responsive Promoter Elements

Zeitschrift für Naturforschung C ◽

10.1515/znc-1999-3-412 ◽

1999 ◽

Vol 54 (3-4) ◽

pp. 220-229 ◽

Cited By ~ 10

Author(s):

Ilka Brehm ◽

Regina Preisig-Müller ◽

Helmut Kindl

Keyword(s):

Cell Wall ◽

Transient Expression ◽

Expression System ◽

Transient Gene Expression ◽

Stilbene Synthase ◽

Fungal Cell Wall ◽

Cauliflower Mosaic Virus ◽

Fungal Cell ◽

Stilbene Synthase Gene ◽

Rna Promoter

Abstract A method for preparing elicitor-responsive protoplasts from grapevine cells kept in suspension culture was established. The protoplasts were employed in order to perform transient gene expression experiments produced by externally added plasmids. Using the gene coding for bacterial β-glucuronidase as the reporter gene, the transient expression under the control of various promoters of stilbene synthase genes were analyzed. The elicitor-responsiveness of promoters from grapevine genes and heterologous promoters were assayed: the grapevine stilbene synthase gene VST-1 and pine stilbene synthase genes PST-1, PST-2 and PST-3. Compared to the expression effected by the cauliflower mosaic virus 35S RNA-promoter, the stilbene synthase promoters caused a 2-5-fold increase in GUS-activity. Incubation of transformed protoplasts with fungal cell wall further stimulated the stilbene synthase promoters but not the 35S RNA-promoter. An even more pronounced differentiation between the promoters was observed when cGMP was included in the transient expression assays. Instead of treating transformed protoplasts with fungal cell wall we administered simultaneously cGMP and the plasmid to be tested. The cGMP-responsive increase was (a) specific concerning the nucleotide applied, (b) characteristic of grapevine protoplasts, and (c) not seen with shortened promoter-GUS constructs or GUS under the control of the 35S RNA-promoter. The highest cGMP-dependent reponse to stress was shown by the promoter of the grapevine stilbene synthase gene VST-1.

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Transient Expression of β-Glucuronidase (GUS) Activity in Embryogenic Callus Tissue of Alkaligrass (Puccinellia distans) using Particle Bombardment

HortScience ◽

10.21273/hortsci.30.4.913a ◽

1995 ◽

Vol 30 (4) ◽

pp. 913A-913

Author(s):

Aaron Brown ◽

Harrison G. Hughes

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Particle Bombardment ◽

Callus Induction ◽

Embryogenic Callus ◽

Callus Tissue ◽

Transient Gene Expression ◽

Plant Cells ◽

Gus Activity ◽

Puccinellia Distans

Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.

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